1. Field of the Invention
The invention relates to the field of post-translational modifications of protein structure. In particular, it relates to the field of assays for one such modification that is involved in the modulation of protein function.
2. Background of the Prior Art
NO donors and endogenously produced NO exert pleiotropic effects including smooth muscle relaxation, cellular proliferation, apoptosis, neurotransmitter release, neurotoxicity, and differentiation. In mediating vasorelaxation, NO stimulates cGMP formation by binding to heme at the active site of soluble guanylyl cyclase which leads to a conformational alteration that augments enzyme activity1. NO also can interact with cysteines to form nitrosothiol adducts2-4, altering the activity of proteins including H-ras5, the olfactory cyclic nucleotide-gated channel6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH)7. Nitrosothiols are exceptionally labile due to their reactivity with intracellular reducing agents such as ascorbic acid and glutathione8, as well as reduced metal ions, especially Cu(I)9, with tissue half-lives of seconds to a few minutes8.
The reversible regulation of protein function by S-hitrosylation has led to suggestions that nitrosothiols function as posttranslational modifications analogous to phosphorylation or acetylation2. The bulk of the evidence for protein regulation by S-nitrosylation has relied on in vitro experiments with NO donors, which in some cases also release other reactive oxygen species, or release NO molecules that differ in electronic structure from NO formed by NOS10. Additionally, the cytoplasm contains high concentrations of glutathione and metals which can bind NO, making it unclear whether S-nitrosylation can be elicited by endogenously produced NO. Despite these difficulties, Stamler and associates have provided evidence that certain proteins, including the ryanodine receptor11, caspase-312, and albumin13, possess nitrosothiol moieties in their endogenous state. Unfortunately, the photolytic-chemiluminescence technique employed in these studies13 was complex precluding widespread application, and it was necessary to purify candidate proteins before assessing their S-nitrosylation state. Thus there is a need in the art for new techniques for measuring this regulatorily important protein modification.